Sterile protection against malaria infection can be induced by multiple exposures to radiation-attenuated sporozoite (RAS) parasite forms in mice and humans, if the RAS remain sufficiently viable to invade hepatocytes. Manufacturing of RAS has several technical hurdles to overcome to allow mass immunization, and therefore, subunit vaccines have been the primary focus of development in recent decades. RTS,S based on Pf circumsporozoite protein (PfCSP), the predominant sporozoite surface antigen, is the most advanced subunit candidate, but has shown only limited efficacy against malaria episodes in Phase III testing. Thus, new subunit vaccine strategies are needed. CSP tolerant transgenic mice are also protected after RAS immunization, implicating additional pre-erythrocytic antigens as targets of sterile immunity. We therefore sought to identify novel candidate pre-erythrocytic vaccine antigens (PEVA) that could add to the level of protection achieved with CSP immunogens alone. We assume that PEVA candidates are transcribed during liver stage (LS) development, and have used transcriptomic data developed in our lab to identify such candidate antigens. LMIV has assessed the protective efficacy of some of these immunogens by DNA vaccination in rodent models of malaria. We highlight findings described in our FY18 publications here: Identification of a Novel CD8 T Cell Epitope Derived from Plasmodium berghei Protective Liver-Stage Antigen We recently identified novel Plasmodium berghei (Pb) liver stage (LS) genes that as DNA vaccines significantly reduce Pb LS parasite burden (LPB) in C57Bl/6 (B6) mice through a mechanism mediated, in part, by CD8 T cells. In this study, we sought to determine fine antigen (Ag) specificities of CD8 T cells that target LS malaria parasites. Guided by algorithms for predicting MHC class I-restricted epitopes, we ranked sequences of 32 Pb LS Ags and selected 400 peptides restricted by mouse H-2Kb and H-2Dballeles for analysis in the high-throughput method of caged MHC class I-tetramer technology. We identified a 9-mer H-2Kb restricted CD8 T cell epitope, Kb-17, which specifically recognized and activated CD8 T cell responses in B6 mice immunized with Pb radiation-attenuated sporozoites (RAS) and challenged with infectious sporozoites (spz). The Kb-17 peptide is derived from the recently described novel protective Pb LS Ag, PBANKA_1031000 (MIF4G-like protein). Notably, immunization with the Kb-17 epitope delivered in the form of a minigene in the adenovirus serotype 5 vector reduced LPB in mice infected with spz. On the basis of our results, Kb-17 peptide was available for CD8 T cell activation and recall following immunization with Pb RAS and challenge with infectious spz. The identification of a novel MHC class I-restricted epitope from the protective Pb LS Ag, MIF4G-like protein, is crucial for advancing our understanding of immune responses to Plasmodium and by extension, toward vaccine development against malaria. Gamma Delta T Cells Are Required for the Induction of Sterile Immunity during Irradiated Sporozoite Vaccinations Whole-sporozoite vaccines confer sterilizing immunity to malaria-naive individuals by unknown mechanisms. In the first PfSPZ Vaccine trial ever in a malaria-endemic population, Vd2 gd T cells were significantly elevated and Vg9/Vd2 transcripts ranked as the most upregulated in vaccinees who were protected from Plasmodium falciparum infection. In a mouse model, absence of gd T cells during vaccination impaired protective CD8 T cell responses and ablated sterile protection. gd T cells were not required for circumsporozoite proteinspecific Ab responses, and gd T cell depletion before infectious challenge did not ablate protection. gd T cells alone were insufficient to induce protection and required the presence of CD8a+ dendritic cells. In the absence of gd T cells, CD8a+ dendritic cells did not accumulate in the livers of vaccinated mice. Altogether, our results show that gd T cells were essential for the induction of sterile immunity during whole-organism vaccination. In addition to these published studies, we made in advances that have not yet been published: Eight of the PEVA candidates identified from previous antigen discovery efforts using transcriptomic data have been examined side by side with five PEVA candidates identified by GenVec, a local Biotech company. All the antigens were combined with CSP for immunization to examine their ability to enhance CSP-mediated protection. Both DNA prime, Ad5 boost and protein prime, Ad5 boost immunization regiments were tested, followed by challenge with sporozoites to examine the sterile protective ability of the CSP combinations. The DNA prime, Ad5 boost study with the thirteen PEVA antigens mentioned above showed that four of the PEVA antigens in combination with CSP displayed higher sterile protection compared to CSP alone. Eight of the PEVA antigens were also expressed and purified from either E. coli or insect cells, and were used in mice immunization studies with CSP. From the results available for four of the proteins, it could be seen that one of the proteins PySHMT combined with PyCSP had higher sterile protection compared to PyCSP alone. Interestingly, the same protein increased the protection shown by CSP in the DNA immunization study as well. Mice immunized with the second set of four proteins in combination with CSP are currently being challenged with P. yoelii sporozoites to determine whether these proteins enhance the protection shown by CSP alone. In FY2018, mice were immunized with CSP alone or CSP plus one of 8 different purified PyPEVA proteins. Seven of the 8 proteins improved upon sterile protection compared to CSP alone. Multiple studies confirmed the reproducibility of this result. In FY2018, PyCSP was used to immunize thicket rats, and confirmed that CSP vaccination of thicket rats offers a model for partial protection. This model can be used to assess novel antigens that are combined with CSP in order to enhance protection.